Cell & Tissue Culture

Research/ medical purposesStudy cell processes e.g.

Cancer Development drug mechanisms, disease processes

Diagnoses e.g. Downs Syndrome, genetic disorders

Skin graftsStem Cells

Stem Cells

Stem cells are unspecialised

Stem cells are able to divide and differentiate

They will in the future be used to “repair” damage in the body.

Commercial applications

PharmaceuticalsAntibiotics (fungi)Human proteins (hGH, Insulin)Enzymes (streptokinase)Complex molecules (cyclosporin)

Steroids (Mexican Yam + bacterium or fungus) Pill, cortisone (anti-inflammatory - $200 $0.46 /g),

Commercial applications

FoodPruteeen (Methylophilus methylotropus)

Animal feed grown on methane gas/ methanolQuorn

fungus Fusarium graminearum)grown on glucose

Autolysed yeast (meat/cheese flavours)Dairy products/BrewingRennin in cheese making

Commercial applications

Industrial manufactureVitaminsPlastics (biodegradable, PHBV)Washing powders Mining (Copper, Gold)Enzyme production (food technology etc.)

Commercial applications

AgricultureGM crops (Flavr savr Tomato)Virus free strawberriesOrchid productionMonsanto Roundup resistant maizeBST for milk production

Commercial applications

BiodegradationOil slicks Organochlorines (PCBs, PCPs)Sewage TreatmentSewage contamination analyses

General Requirements for cell culture

Growth requirements nutrient medium

heterotroph, photoautotroph (light), chemoautotroph surface on which to grow growth factors

Temperature Hyperthermophiles, thermophiles, mesophiles, psychrophiles

pH (buffered) acidophiles,

Prevent contamination (aseptic technique, antibiotic, fungicide)

Bacterial Growth Media

Most bacteria are heterotrophs Require complex medium (defined)

carbon source (glucose), nitrogen source (ammonium salts), energy source (glucose), micronutrients (Fe, Co, Mn etc)

Often undefined e.g Yeast extract, peptone, casein hydrolysate

Vitamins and specific amino acids may also be included Special media contain specific requirements e.g.

Blood/ milk/ acid pH etc.

Growth conditions

Oxygen requirementsObligate aerobes

Absolutely require oxygen Large scale cultures need to aerate

Facultative aerobes Can grow without oxygen, but grow better with it

Obligate anaerobes Oxygen is toxic to them Grown in anaerobic jars (filled hydrogen & carbon

dioxide)

Preventing contamination

Aseptic technique

Selective media (contain antibiotics or specific nutrients/ pH)Antibiotics (selective) Fungicides

Containment (cabinets)

Sterile media (autoclave/ 0.2m filter)

Measuring Growth Rate

Haemocytometer

Flow cytometer

Plate dilution method (viable count)

Colorimetry (densitometry)

Growth curves

Slow growth phase Bacteria preparing for cell division (also low numbers)

Logarithmic (exponential growth phase) Unlimited growth rate

Plenty food, space Wastes not yet at toxic levels

Stationary (declining) phase Limited by food, space Toxic build up

Continuous culture Refresh part of culture

Mammalian Cell Culture

More difficult than bacteriaconditions more carefully regulatedMedia more complex (amino acids, vitamins)pH indicator (phenol red – indicates CO2)Sera (e.g. foetal calf serum / donor horse

serum - contain growth factors) If cells divide, they usually die after a finite

number of divisions.

Mammalian Cell Culture

Primary culture Culture cells taken from an animal Limited life span More like reality

Treat with enzymes to disrupt cells Trypsin / collagenase

Surface attachment Cells are anchorage dependent Reach confluence Subculture

Mammalian cell CultureContinuous cell lines

Derived from tumours (HeLa, PC12) Transformed (viruses) - lost cell cycle control Immortalised

Easier to grow routinely Continue to divide provided correct conditions maintained Subculturing required when confluent (covering the entire plate Risk of cell line changing (mutating) Can lose anchorage dependence

Cloning Allows isolation of single cells Novel mutants

Plant cell culture

Simpler requirements than animal cells.Easier to produce a whole plant from single cell Nuclear totipotency – capable of producing all

differentiated cell types because genome contains all genes (all cells are nuclear totipotent – in theory - DtS).

Explants (cells or pieces of tissue) grown in appropriate media (light required – photoautotroph)Growth regulators (plant hormones) induce differentiation to produce whole plantsProtoplasts can also be grown into whole plants – to produce hybrids or genetically modified cells.Lack of cell wall means genes are easily introduced

Method